FISSION YEAST SCHIZOSACCHAROMYCES POMBE AS A MODEL … · pombe by transmission electron microscopy (TEM) require transformation of the living cells from their native, hydrated state

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FISSION YEAST SCHIZOSACCHAROMYCES POMBE AS A MODEL SYSTEM FOR ULTRASTRUCTURAL

INVESTIGATIONS USING TRANSMISSION ELECTRON MICROSCOPY

Hana Ďúranová1*, Miroslava Požgajová1, Marta Novotová2, Norbert Lukáč3, Zuzana Kňažická4

Address(es): 1Slovak University of Agriculture in Nitra, AgroBioTech Research Centre, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic.

2Biomedical Research Center, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05 Bratislava, Slovak Republic.

3Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Animal Physiology, Tr. A. Hlinku 2, 949 76 Nitra, Slovak

Republic. 4Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic.

*Corresponding author: [email protected]

ABSTRACT

Keywords: Schizosaccharomyces pombe; model system; ultrastructure; transmission electron microscopy; sample preparation

INTRODUCTION

In the most common terms, a model organism is defined as a non-human

biological subject used in scientific laboratories to investigate specific biological

processes and mechanisms under physiological or experimental conditions. In

this regard, it is believed that findings and theories made in the model organism

can be applied to higher species that are expected to be more complex than the

model system (Ankeny and Leonelli, 2011). According to Al-Wattar (2017),

the criteria for a model biological system are as follows: (i) the used organism

should be easy to obtain, grow and manipulate; (ii) its operation size should be

convenient; (iii) it should be inexpensive to maintain; (iv) the model organism

should have short life cycle; (v) the organism can be genetically modified, and

(vi) it should have the potential to deliver economically important results.

GENERAL CHARACTERISTICS OF MODEL SYSTEM

SCHIZOSACCHAROMYCES POMBE

Yeasts are single-celled (unicellular) eukaryotic organisms characterized by easy

manipulation, low-cost cultivation demands, and short life cycle allowing

researchers to study fundamental biological properties. They are harmless,

genetically amendable with one of the smallest genomes permitting whole-

genome characterization of various cell processes (Zhao, 2017). Additionally,

they possess basically the same subcellular structure as cells of higher organisms

(animals and plants) venturing their use as a laboratory tool for basic and applied

spheres of biology, life science, medicine, or biotechnology (Osumi, 2012).

Schizosaccharomyces pombe (S. pombe), often known simply as “fission yeast”

is a unicellular eukaryote belonging to the Ascomycetes which are characterized

by formation of sexual spores inside an ascus (Latin for “bag”; Hoffman et al.,

2015). Saare and colleagues isolated the fission yeast from a millet beer which

was contaminated due to its delaying journey from East Africa to Europe

(Germany). Isolation of the pure S. pombe culture followed by its first detailed

description was done in early 1890s by Ziedler and his supervisor Paul Lindner

(Hayles and Nurse, 2018). In general, pombe is the Swahili word for “beer” (or

at least a beer-like fermented beverage), and the fission yeast is used for its

fermentation (Hoffman et al., 2015). Besides this, S. pombe often occurs within

the process of molasses fermentations during preparation of distilled

commodities such as rum, arrak, tequila or cachaca (Gomes et al., 2002) and is

known for its ability to utilize malic acid resulting in elimination of undesirable

acidity in wine (Volschenk et al., 2003; Benito et al., 2016).

From morphological point of view, the fission yeast cell (Figure 1) has a

cylindrical rod shape of 4 μm in diameter (Piel and Tran, 2009) lined by a cell

wall which mainly consists of polysaccharides directly responsible for cell wall

rigidity (Grün et al., 2004). In 1977, Manners and Meyer (Manners and Meyer,

1977) determined the exact composition of the cell wall as follow: 9 - 14 % α-

galactomannan, 18 - 28 % α-1,3-glucan, 42 % β-1,3 glucan and 2 % β-1,6-

glucan. The exoskeletal rigid structure (representing ~20 % of the cell dry mass)

is required for survival of S. pombe due to its protective efficacy against cell

destruction caused by an increased internal turgor pressure or external

mechanical injuries. Deeper understanding of the cell wall structure and

composition will bring a valuable insight in S. pombe morphogenetic

specifications, as the cell wall also defines the shape of the cells (Pérez and

Ribas, 2017). Moreover, the protoplasts of the fission yeast are able to fully

restore cell walls in liquid media, thereby reverting to normal cells. This feature

of the regenerating process makes S. pombe a useful tool to analyze a process of

de novo synthesis of the cell wall (Osumi, 2012).

Figure 1 Representative microphotograph of S. pombe cells

The unicellular fission yeast Schizosaccharomyces pombe (S. pombe) has become a prominent model system to elucidate a various

range of biological processes which are highly conserved in mammalian cells. Ultrastructure of the cells related to the organelle

morphology is useful in the generation of the comprehensive overview of the cell function. Transmission electron microscopy provides

a unique tool to study cell architecture under physiological conditions, as well as ultrastructural changes of the cells due to toxic or

beneficial effects of diverse additives. In recent years, S. pombe has also proved to be a suitable cell system for transmission electron

microscopy investigations. In the current study, general features of S. pombe are described. In addition, conventional specimen

preparation technique and the important discoveries of cell architecture emerging from transmission electron microscopy studies of S.

pombe are summarized.

ARTICLE INFO

Received 1. 4. 2019

Revised 21. 6. 2019

Accepted 24. 6. 2019

Published 1. 8. 2019

Review

doi: 10.15414/jmbfs.2019.9.1.160-165

J Microbiol Biotech Food Sci / Duranova et al. 2019 : 9 (1) 160-165

161

Visualized are bright field images of vegetative wild type cells under inverted

DM IL LED Leica microscope, equiped with Leica EC3 digital color camera,

magnification 40x.

The growth of fission yeast cells is commonly established through the cell ends

elongation in a linear polarized fashion ensuring their typical cell morphology

(Grün et al., 2004). Schizosaccharomyces pombe grows by elongation from 7 to

14 μm in length (Piel and Tran, 2009). During mitotic cell division, the cell

growth is ceased. Hence, the cell length serves as a sensitive cell cycle phase

indicator of the analyzed cells, and additionally, cell size monitoring facilitates an

evaluation of the timing of mitotic entry (Forsburg and Rhind, 2006; Hagan et

al., 2016). Compared to other eukaryotic species, S. pombe grows quickly. Under

normal conditions, a wild-type cell takes about 2.5–3 h to complete a cell cycle

which is similar to that of other eukaryotes (i.e., it also includes G1, S, G2 and M

phases; Gómez and Forsburg, 2004). Schizosaccharomyces cells divide by

medial fission (Hayles and Nurse, 2018) followed by cleavage of the primary

septum (Peckys et al., 2011). This cell division is morphologically very

symmetric and thus results in a formation of two almost identic daughter cells

(Lin and Austriaco, 2014). Besides asexual multiplying through the mitotic

cycle, the cells can mate and form diploid zygotes. Sporulation immediately

follows meiosis resulting in the production of four round or oval haploid

ascospores enclosed within an ascus (Zhao, 2017). Mating in S. pombe occurs

under conditions of nutritional stress (Martín et al., 2013). Differentiation into a

variety of filamentous or hyphal growth forms is also feasible in the fission

yeasts (Amoah-Buahin et al., 2005).

Regarding genetic features, the genome of S. pombe was fully sequenced in 2002,

becoming the sixth model eukaryote with entire sequenced genome (following

Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster,

Arabidopsis thaliana, and Homo sapiens; Yanagida, 2002). The fission yeast has

the smallest sequenced eukaryotic genome (13.8 Mb; Wood et al., 2002)

organized into only three chromosomes of 5.6, 4.8 and 3.6 Mb, respectively

(Hayles and Nurse, 2018). It has single copy genes making it advantageous for

gene functional studies. Moreover, its large repetitive centromeres (40–100 kb)

are similar to those of mammals (Roque and Antony, 2010).

The unfussiness in growing, amenability to genetic manipulations, and regular

cell size have made S. pombe a popular model organism for the study of number

major topics in cell biology such as cell cycle (Hagan et al., 2016), cell division

(Piel and Tran, 2009), chromosome segregation (Kovacikova et al., 2013;

Pozgajova et al., 2013; Yamashita et al., 2017), cell growth conditions

(Petersen and Russell, 2016), cell morphology (Huang et al., 2003), cell wall

biosynthesis (Leon et al., 2013), the cytoskeleton (Kovar et al., 2011), and many

others.

CONVENTIONAL PREPARATION OF S. POMBE FOR TRANSMISSION

ELECTRON MICROSCOPY

In general, ultrastructural studies of various biological samples including S.

pombe by transmission electron microscopy (TEM) require transformation of the

living cells from their native, hydrated state to a dry one that reliably maintains

the structural and biological relationships. Four basic conditions must be

complied during sample preparation: (i) to prevent destruction of the cell

structures and organelles due to vacuum in the electron microscope; (ii) to

generate ultra-thin sections in order to eliminate multiple electron scattering (iii);

to minimize structural alterations caused by the electron beam; (iv) to maximize

the contrast in the object-defined resolution (Slayter and Slayter, 1992;

Böttcher, 2012). Taken together, the main aim of the sample processing for TEM

is to ensure production of sufficiently thin sections that allow transmission of

electron beam through the sample to obtain an image. Hence, construction of thin

sections requires three main approaches of sample preparation: fixation in the

first step, followed by dehydration, and finished by infiltration/embedding

(Wright, 2000).

Growth conditions and harvesting of S. pombe

The difficulty of the yeast cell processing for TEM analysis compared to higher

eukaryotic cells lies in the presence of the thick cell wall acting as an obstacle

against fixative diffusion (Bauer et al., 2001). To minimize this refractory

feature of the S. pombe cells to preservation, it is necessary to precisely regulate

culture conditions of yeasts. Indeed, recommended timing for fixation of yeast

cells is the early log phase which is characterized by minimal density of the cell

cytoplasm and maximal cell wall permeability (Wright, 2000; Sipiczki, 2016).

In this phase, cell density is limited to 5 x 106 / ml (i.e., OD600 = 0.5). On the other

hand, cells in the stationary phase possess large, electron-dense vacuoles and

numerous electron-dense inclusion bodies that obscure ultrastructural details

(Wright and Rine, 1989). To ensure that cells are really growing exponentially,

it is recommended that cultures have been maintained at a low density (< 0.5) for

three or more generations (Mulholland and Botstein, 2002).

The average life cycle of S. pombe lasts 2 – 4 hours at 30 °C which represents

optimal growth temperature in the laboratory. Standard and the most widely used

culture medium is rich medium (Standard Yeast Extract with Supplements - YES

medium) containing all nutrients such as yeast extract and glucose in excessive

amounts, and specific amino acids or nucleobases. This medium with no addition

of antibiotics is normally used for yeast growth without selection (Petersen and

Russell, 2016; Zhao, 2017).

During cell harvesting it is essential to keep cells as intact as possible forming a

compact firm mass (Sipiczki, 2016). In the case of fission yeasts, cell isolations

by centrifugation (Bauer et al., 2001; Roque and Antony, 2010; Sipiczki, 2016;

Koch et al., 2017) or filtration (Petersen and Russell, 2016; Giddings et al.,

2017) appear to be the most preferred methods. However, both of them can cause

damages to the cells resulting in structural changes. Centrifugation, the most

commonly used process of cell harvesting, leads to cytoskeleton and nuclear

positioning (Roque and Antony, 2010). Therefore, centrifugation of yeast cells

should be carried out after the cells have been chemically fixed. Taking into

account this consideration, the first fixative reagent is added as 2x stock to the

medium with the cells (1:1 ratio) and incubated for 5 minutes at room

temperature. Subsequently, the cells are gently centrifuged and resuspended in

fresh 1x stock fixative solution to finalize cell preservation as it was previously

described by Wright (2000) and Roque and Antony (2010).

Fixation

Fixation is the first step providing the preservation of the cells in a state as

similar as possible to their vital state. It means that fixation must halt potentially

destructive autolytic processes to protect cells against disruption during their

embedding and sectioning, as well as to ensure morphological stability of the

cells exposed to electron beam (Bozzola and Russell, 1999). Generally, three

possible approaches of the fission yeast processing are usually used to retain

normal structural organization of the cells for successful staining procedures. The

first and the most commonly used technique is the fixation by cross-linking.

Alternatively, fixation by precipitation or fixation by freezing (i.e., cryofixation)

can also be applied (Sipiczki, 2016). Due to requirement of special laboratory

facilities related to cryofixation which are often costly and therefore unavailable

in many laboratories, the most widely used technique of cell preservation is based

on cell treatment with chemicals known as fixatives. Their task is to remove the

water content from cell structures composed of proteins, lipids and

carbohydrates, thereby enabling stronger connection between molecules

responsible for construction of these structures (Özen and Ceylan, 2013).

For ultrastructural analysis, the most popular method of fixation is double

fixation in an aldehyde (primary fixation; sometimes called prefixation) followed

by secondary (post) fixation in oxidizing agent (Wright, 2000; Mielańczyk et

al., 2015; Ayub et al., 2017).

Primary fixation (Prefixation)

Currently, the monofunctional and bifunctional aldehydes, i.e., formaldehyde

(HCHO) and glutaraldehyde (CHO-CH2-CH2-CH2-CHO), are the most widely

and successfully used reagents for primary fixation. These compounds react with

amino groups of proteins and macromolecules associated with proteins (e.g.,

histoproteins associated with DNA or lipoproteins; Bozzola, 2007) to stabilize

them by forming inter- and intra-chain crosslinks. However, they demonstrate

little, if any, reactivity with lipids (Wright and Rine, 1989).

Having two aldehyde groups at both ends of the molecule, glutaraldehyde (GA)

binds strongly proteins (especially those containing amino acids such as lysine,

tyrosine, tryptophan, and phenylalanine; Park et al., 2016) causing their

irreversible cross-linking. Since GA contains five carbons in its molecular

structure and is also uncharged in a solution, its penetration through the cell wall

of the yeasts is very slow (Frankl et al., 2015). Usually, it is available in various

concentrations ranging from 8 to 70 %. For cell fixation, the concentrations from

0.25 to 3 % are commonly used (Wright, 2000). According to many authors

(Bauer et al., 2001; Roque and Antony, 2010; Asakawa et al., 2012; Sipiczki,

2016), GA at a concentration of 2 % is extensively employed for yeasts as a

prefixative.

Formaldehyde (FA) preserves the structures of the cells via formation of Schiff

base intermediates with free amino groups of proteins, thereby stabilizing

adjacent proteins (Perkins and McCaffery, 2007). It is typically used at final

concentrations ranging from 0.5 to 4 % (Wright, 2000). For electron microscopy,

powdered polymerized formaldehyde, i.e., paraformaldehyde (PFA) is used. The

small size of PFA and only one aldehyde group permit its rapid diffusion across

the cell wall of yeasts (Frankl et al., 2015). However, in spite of deeper and

faster penetration, it has a relatively weaker (especially in low concentrations)

reversible fixation power as compared to GA (Hajibagheri et al., 1999; Park et

al., 2016).

In recent years, GA has begun to be used in conjugation with FA known as

Karnovsky's fixative (Karnovsky, 1964). Karnovsky's original fixative

(containing 5 % GA and 4 % FA) is often modified to yield fixatives with lower

concentrations of both FA and GA (e.g., 2 % PFA and 2.5 % GA; Bozzola and

Russell, 1999). It is usually employed in difficult to fix biological samples, as

well as in those for which a preservation protocol is absent (Bozzola, 2007). The

combination of GA with PFA as a fixative for electron microscopy takes

advantage of the rapid diffusion of PFA into the cells initializing the structural

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stabilization whilst thorough cross-linking is guaranteed by more slowly

penetrating GA (Kiernan, 2000; Wright, 2000). For S. pombe, a combination of

0.5 % GA and 3 % PFA in 0.1 M phosphate buffered saline (PBS; pH 7.2) can

also be applied. Unfortunately, PFA can negatively affect fixation of

ultrastructural components within the cell resulting in a potential reduction of the

clarity of several intracellular structures (Sipiczki, 2016).

Although GA and PFA are excellent fixatives for preserving the microstructure

of various cells including S. pombe, they do not possess enough contrast to stain

cell components. Additionally, they have almost no power to fix lipids in

biological membranes thus leading to their extraction during dehydration and

infiltration. As a result of this process, electron transparent spaces are observed

instead of initially localized membranes (Wright, 2000). Therefore, postfixation

with oxidizing compounds should be performed.

Secondary fixation (Postfixation)

The most commonly used postfixatives that preserve membranes of yeasts are

potassium permanganate (KMnO4) and osmium tetroxide (OsO4). Potassium

permanganate has been extensively used as a fixative for EM from 1956 (Luft,

1956). To visualize the ultrastructure of cell membranes and membrane profile of

organelles (such as mitochondria, endoplasmic reticulum, nucleus, vacuole, Golgi

and endosomes), extraction of cytoplasmic components from aldehyde-fixed cells

via incubation with KMnO4 (0.5 to 6 % for 1 to 24 h) is recommended

(Baharaeen and Vishniac, 1982; Wright and Rine, 1989; Lum and Wright,

1995; Tronchin and Bouchara, 2006). By depositing MnO2, the membranes

appear to be also highly contrasted (Sipiczki, 2016). Despite the fact that

permanganate fixation is strongly advised for the investigations of complete

membrane organization, the fixative does not protect several prominent non-

membranous cell components such as microfilaments, microtubules or ribosome

(Frankl et al., 2015). Indeed, all that remains are the electron-dense membrane

profiles in a consistently gray cytoplasm even after longer incubation period

(Wright, 2000). Thus, KMnO4 is sparsely used for internal structure visualization

of yeasts.

Osmium tetroxide reacts strongly with unsaturated fatty acids in lipids (changing

them to stable glycol osmate; Park et al., 2016) to provide good fixation of

membranous structures and the lipids themselves (Wright, 2000). Due to its high

density allowing electron scattering, OsO4 also enhances the contrast of the lipid

bilayers visualizing particular membranous compartments (Frankl et al., 2015).

Besides this, osmium also reacts with denatured nucleic acids and proteins rich in

amino acids with unsaturated double bonds (such as cysteine and tryptophan;

Park et al., 2016). By contrast, carbohydrates are not preserved by the OsO4

postfixation (Bozzola, 2007). According to many authors (Tronchin and

Bouchara, 2006; Osumi, 2012), OsO4 extracts much less material from the cells

leading to a visualization of microtubules, microfilaments, ribosomes and

chromatin as compared to the KMnO4. However, unlike KMnO4, osmium

requires for its good cell penetration either cell wall permeabilization or its

complete removal by enzymatic digestion with lytic enzymes (lyticase,

zymolyase and glusulase; Wright, 2000). Removing the wall not only facilitates

permeation of the embedding resin but also permits the cell to expand slightly,

conferring differences in density that provide variation in visual contrast (Byers

and Goetsch, 1991). The efficiency of cell wall removal depends primarily on

the enzymatic conditions, e.g., the nature of the enzymes, their concentration, and

the time and temperature of digestion (Bauer et al., 2001; Požgajová et al.,

2017). After enzymatic treatment, the cells are washed and subsequently fixed in

1–2 % OsO4 in an appropriate buffer, according to the used protocol for time

points ranging from 15 to 60 minutes at 4 °C or room temperature (Wright,

2000).

During prefixation and postfixation processes, the fixed cells die and the released

contents of the lysosome can often change the pH accelerating destruction of

macromolecules in the cells (Park et al., 2016). To provide optimal cross-linking

and ultimate embedding of the sample, optimal pH should be maintained by the

delivery of prefixatives/postfixatives in buffer solutions. Phosphate, cacodylate or

organic based buffers such as PIPES are the most frequently applied. Buffers

used for cultivation of yeast cells are typically adjusted to a slightly acidic pH. In

addition, they are often supplemented with mineral compounds such as MgCl2,

CaCl2, or EGTA (Wright, 2000). For instance, calcium ions added to PIPES and

cacodylate buffers are able to improve membrane preservation (Frankl et al.,

2015). Although buffers on phosphate and cacodylate bases are the most

commonly employed, it is highly advised to consider the use of organic buffers as

a substitution due to their non-destructive effects on fine cell structures and their

non-toxicity (Bozzola, 2007).

En Bloc staining

After complete fixation and several washing steps aimed to remove the fixative

residues, cells are treated with 1–2 % aqueous uranyl acetate at room temperature

for 30–240 minutes (Wright, 2000) to provide a general background contrast to

the sample. Since the contrast in the TEM primarily depends on the electron

density differences of the organic molecules in the cells which are usually present

in insufficient quantities in fixed samples, the specimens are frequently

“poststained” with reagents attaching metals of high atomic mass to the cell

structures (such as uranium; Sipiczki, 2016). Indeed, uranyl acetate reacts

strongly with phosphate and amino groups leading to staining of nucleic acids

and phospholipids in membranes (Kuo, 2007). In addition, uranyl acetate

provides some degree of fixation without a major effect on protein conformation

(Roque and Antony, 2010).

Dehydration

Dehydration is the process by which the free water in the fixed sample is replaced

by an organic solvent (Winey et al., 2014). This step is necessary due to water

immiscible nature of resins that are used for infiltration and embedding of the

samples thus preparing them for the process of sectioning (Sipiczki, 2016). For

this purpose, cells are dehydrated by incubation in ascending rates of ethanol (or

acetone) concentrations (25, 50, 75, 95, and 100 %) for 5 minutes (or less) in

each solution (Wright, 2000). Prior to acetone, ethanol is preferred because it is a

more powerful lipid extractor within the cells. Moreover, anhydrous acetone is

able to absorb atmospheric water, and it is also efficient radical scavenger,

thereby suppressing block polymerization of embedding samples (Sipiczki,

2016).

Infiltration, Embedding and Polymerization (Hardening)

Embedding is the process of sample infiltration with resins that can be

polymerized into a hard plastic material suitable for thin sectioning (Winey et al.,

2014). Since ethanol in dehydrated cells is not miscible with embedding media,

its replacement by another intermediary solvent with high miscibility with the

embedding medium is essential. As a standard transition solvent reducing the

embedding media viscosity and thus improving its infiltration into the samples,

propylene oxide (PO; 1,2-epoxypropane) is usually used (Sipiczki, 2016). It is

common to pass cells for a few minutes through the pure PO prior to infiltration.

Then, the cells are pre-infiltrated with various proportions of PO and the

embedding medium and after that with the pure embedding medium (Mascorro

and Bozzola, 2007).

Two main categories of plastic resins are available for yeast TEM, i.e., epoxy

(Araldite, Epon, Spurr’s resin) and acrylic (Lowicryl, LR White, LR Gold) resins

(Wright, 2000; Frankl et al., 2015; Sipiczki, 2016). Epoxy resins are used for

analysis of the cell morphology while acrylic-based resins better preserve the

antigenicity of biological samples (Frankl et al., 2015). Epoxy resins can be

extensively crosslinked with no impact on their plasticity allowing preparation of

the sections as thin as 50–60 nm (Roque and Antony, 2010), and they also

possess excellent poststaining properties (Winey et al., 2014). The resins

polymerize at 60 °C for 24 hours before their ultra-thin sectioning by

ultramicrotome (Wright, 2000). On the other hand, the hydrophobic feature of

the LR White resin permits the penetration of aqueous solutions containing

special antibodies. It can be hardened either by addition of accelerators, heat

(Sipiczki, 2016), or UV light which allows its polymerization at very low

temperatures (-45 or -50 °C; Roque and Antony, 2010).

Thin-sectioning and Staining

After polymerization, thin sections are cut and mounted on grids for observation

in TEM. Generally, thin sectioning (ultramicrotomy) is difficult to acquire

sufficient skills. It involves: (i) trimming or shaping the specimen block, (ii)

preparing ultramicrotome knives and specimen support grids, (iii) cutting

sections on the ultramicrotome, (iv) transferring the sections onto the specimen

grid, and (v) staining them to enhance contrast (Bozzola and Russell, 1999).

First of all, the block should be trimmed in the way that the section is wide in the

parallel axis to the diamond knife and short in the perpendicular axis to the knife.

This shape will increase the number of sections per ribbon and allows the search

of the same cell across the sections (Roque and Antony, 2010). Once trimmed,

the block is mounted in the ultramicrotome that cuts precisely controlled slices

over a diamond (or glass) knife edge to produce sections of ∼60–80 nm thickness

(Winey et al., 2014). Afterwards, sections floating on the surface of water

contained in the trough of the knife are picked on a copper screen mesh, or grid,

and stained for contrast using salts of a heavy metal prior to visualization under

TEM (Bozzola and Russell, 1999). En bloc stained samples require to be stained

only with lead citrate which has the ability to interact with negatively charged

components (e.g., hydroxyl groups) or areas reacting to OsO4 (e.g., membranes;

Sato, 1968). Since the phosphate buffers usually increase the intensity of

staining, lead citrate can also stain the constituents with phosphate groups (Kuo,

2007). After all of the processing steps, dried grids are visualized under TEM.

TRANSMISSION ELECTRON MICROSCOPY AS A UNIQUE

TECHNIQUE IN ULTRASTRUCTURAL STUDIES OF

SCHIZOSACCHAROMYCES POMBE

Generally, TEM investigations are necessary for understanding various biological

processes (e.g., formation and fusion of vesicles during secretion), cytoskeleton

organization, precise mechanisms of biogenesis and degradation of cellular

J Microbiol Biotech Food Sci / Duranova et al. 2019 : 9 (1) 160-165

163

organelles, as well as specific localization of certain gene products (Wright,

2000). To ensure maintenance of all cellular requirements, organelles as dynamic

and flexible structures are capable of changing their shape and size. Many of

these modifications can be observed during normal cell cycle (mitosis, meiosis)

or as a part of adaptive mechanism of the cells against environmental stress

reflecting alterations in organelle functions (Heald and Cohen-Fix, 2014).

The cell of S. pombe contains a minimal number of molecular compounds which

is displayed by lower number of subcellular elements (Roque and Antony,

2010). This advantage makes the fission yeast to be a convenient model organism

to study the cell ultrastructure using TEM under normal or experimental

conditions. The architecture of S. pombe was first studied by TEM using KMnO4

and OsO4 as fixatives in 1964 (Maclean, 1964). The serial sectioned TEM

images showed 0.1-0.2 µm thick cell wall, cell membrane, central nucleus (2-3

µm in diameter) with granular nucleolus, bounded by double nuclear membrane,

and various cytoplasmic structures (vacuoles, membranes and vesicles, and

granules). Later, detailed ultrastructure of the cell wall in the S. pombe was

performed by several researchers (Kopecká et al., 1995; Osumi et al., 1998;

Humbel et al., 2001; Sugawara et al., 2003). The cell wall has been recognized

as a triple-layered structure involving two electron dense layers (outer one and a

layer bordering the cell membrane) separated by an adjacent less dense layer with

fine branched filamentous structures. In the fission yeast, TEM has also been

used as a unique tool for examination of mitotic and meiotic events. First

visualization of mitosis in S. pombe using TEM was performed by McCully and

Robinow (1971). In this study, the presence of disk-shaped electron-dense

organelles, specifically called as kinetochore equivalents (KCEs) was revealed.

Further studies with S. pombe have utilized TEM to visualize the septum of

dividing cells (Humbel et al., 2001), nuclear division during mitosis (Tanaka

and Kanbe, 1986), fusion and erosion of cell walls during conjugation (Calleja

et al., 1977), nuclear envelope during first and second anaphase (Asakawa et al.,

2010), ascospore formation (Yoo et al., 1990; Asakawa et al., 2001),

ultrastructural alterations in mitochondria morphology during division of the

organelle (Osumi and Sando, 1969), dynamics of cytoplasmic structures

(vesicles, filasomes, microfilament, and microfilament-associated granules,

dictyosomes) in the cell cycle (Kanbe et al., 1989), and dynamics of cell

membrane and/or cellular structures (mainly the Golgi apparatus and the

secretory vesicles) during de novo synthesis of cell wall in reverting protoplast

(Naito et al., 1991; Osumi et al., 1998; Takagi et al., 2003).

Moreover, TEM has also been used in ultrastructural studies of S. pombe under

defined environmental conditions. In the study by Amoah-Buahin et al. (2005),

the ultrastructure of hyphal growth forms of wild-type S. pombe initiated in a

consequence of nitrogen starvation was investigated. Ayscough et al. (1993)

have reported damage of the microtubular system leading to separation of Golgi

apparatus cisternae in S. pombe treated with anti-microtubule agent thianedazole.

Following this study, S. pombe may be used as a unique model for study of

ultrastructural changes in the cells due to toxic impact of various substances.

Moreover, since it shares similarities with many fundamental biological

processes occurring in higher eukaryotic organisms (e.g., mRNA splicing, post-

translational modifications as N-glycosylation protein, cell-cycle regulation,

cAMP-PKA pathway, autophagy, nutrient-sensing pathways as the target of

rapamycin – TOR pathway; Rosas-Murrieta et al., 2015), the same

ultrastructural alterations in S. pombe cells induced by various exogenous factors

(such as the impact of environmental contamination) can be expected also in

higher organisms (including humans). Thus, the use of fission yeast S. pombe as a

model organism for ultrastructural studies may bring a new knowledge in the

process, progress or regulation of various biological processes with the direct

application to mammalian cells including human.

In our laboratory, the methods of conventional preparation of S. pombe for TEM

are being introduced. The TEM technique will provide additional information

about ultrastructural changes in the cells due to the effect of environmental stress

(heavy metals). However, our preliminary results (not yet published) indicate that

optimization of the process is still necessary. Indeed, the key step for precise

preservation of cellular structure by fixatives requires a sufficient cell wall

removal by the action of enzymes. The concentration, as well as the incubation

time for zymolyase treatment is now optimized to obtain more relevant

ultrastructure of the S. pombe cells with well-contrasted cell membranes and

compartments.

CONCLUSION

Transmission electron microscopy is a powerful and very precise technique to

study morphological structures such as surface features, shape, size or

ultrastructure of biological samples and their organelles. In general, S. pombe is a

popular model system used for characterization of basic eukaryotic processes at

cellular and molecular levels. Studies performed on a convenient model system

(such as the fission yeast) using TEM allow scientists to analyze cytoskeleton

organization, the mechanisms of organelle biogenesis and degradation, and

ultrastructure and dynamics of organelles as a consequence of adaptive

mechanisms against various exogenous factors. As a unique model system, S.

pombe can also be used in ultrastructural studies evaluated the toxic effects of

environmental pollution on cell ultrastructure. Since it shares many essential

biochemical, molecular and genetic characteristics with higher eukaryotic

organisms, the same ultrastructural alterations found in S. pombe cells might also

be expected in higher organisms (including humans). However, to achieve results

with such noticeable value it is of great importance to lay stress on very accurate

sample preparation. Moreover, the exact data interpretation is essential for correct

definition of obtained results and conclusion presentation.

Altogether, ultrastructural analyses performed with a single-celled eukaryotic

organism have the power to bring new insights to the changes in the behavior of

organisms to various challenges of its living conditions on cellular and

subcellular level that can be to a large extent transformed to higher biological

systems.

Acknowledgments: This research was supported by European Community under

project No. 26220220180: Building Research Centre “AgroBioTech and by the

Scientific Agency of the Slovak Republic VEGA No. 1/0163/18, APVV-15-

0543.

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